Anticoagulant fusion protein anchored to cell membrane

ABSTRACT

The invention relates to the inhibition of blood coagulation, especially during organ rejection, and in particular the inhibition of delayed vascular rejection. The invention provides anticoagulant proteins which are anchored to cell membranes. The anticoagulant function preferably provided by heparin, antithrombin, hirudin, TFPI, tick anticoagulant peptide, or a snake venom factor. These anticoagulant proteins are preferably prevented from being constitutively expressed at the cell surface. In particular, expression at the cell surface is regulated according to cell activation, for instance by targeting the protein to a suitable secretory granule. Expression of these proteins renders cells, tissues and organs less vulnerable to rejection after transplantation (e.g. after xenotransplantation).

FIELD OF THE INVENTION

This invention relates to the inhibition of blood coagulation,especially during organ rejection.

BACKGROUND TO THE INVENTION

The surgical technique of organ transplantation has now beensuccessfully practised for several decades and, because of its success,the procedure has become widespread and, arguably, routine. However, thesupply of suitable transplant organs is not able to match ever-risingdemands. Because of the shortage of suitable human (ie. allogeneic)organs, the possibility of using animal (ie. xenogeneic) organs in humantransplant operations (“xenografting” or “xenotransplantation”) has beenreceiving increased attention in recent years (eg. Nature 1997; 385:285). Porcine donor organs are thought to be suitable candidates becausepigs are anatomically and physiologically similar to humans and are inabundant supply.

Xenografting is currently hindered, however, by the severe andwell-documented problems of rejection. This process can be divided intodistinct stages, the first of which occurs within minutes oftransplantation. This is known as the hyperacute response and is causedby existing antibodies in the recipient which recognise and react withforeign antigens on the endothelial cells (ECs) of the xenograft. Thisrecognition triggers the complement cascade which in turn leads to lysisand death of ECs of the transplant.

This initial hyperacute rejection is then reinforced by the delayedvascular response (also known as acute vascular rejection or delayedxenograft rejection). The lysis and death of ECs during the hyperacuteresponse is accompanied by oedema and the exposure of adventitial cells,which constitutively express tissue factor (TF) on their surface. Tissuefactor is thought to be pivotal in the initiation of the in vivocoagulation cascade, and its exposure to plasma triggers the clottingreactions. Thrombin and TNF-α become localised around the damaged tissueand this induces further synthesis and expression of TF by ECs.

The environment around resting ECs does not favour coagulation. Severalnatural coagulation inhibitors are associated with the extracellularproteoglycans of ECs, such as tissue factor pathway inhibitor,antithrombin III, and thrombomodulin. The recognition of the foreigntissue by xenoreactive natural antibodies (XNAs), however, causes theloss of these molecules.

Together with the exposure and induction of tissue factor, theanticoagulant environment around ECs thus becomes pro-coagulant.

The vascularised regions of the xenograft thus become sites of bloodclots, a characteristic of damaged tissue. Blood flow is impaired andthe transplanted organ becomes ischaemic. A fuller account of delayedvascular rejection can be found in Bach et al. (1996).

The use of xenogeneic organs in transplants is therefore hindered by aninitial hyperacute rejection followed by a prolonged vascular rejection,possibly followed by T-cell mediated rejection. Inhibition of themechanisms responsible for these rejections could facilitate the use ofxenografts.

The simple administration of suitable inhibitors, however, is not aparticularly suitable approach. Completely inhibiting complement in arecipient animal is tantamount to immunosuppression, leaving the subjectprone to opportunistic infections. Similarly, inhibiting the coagulationcascade in a recipient will leave the animal susceptible to uncontrolledpost-operative bleeding. Therefore the inhibitors should desirably belocalised in the recipient to the site of the xenograft.

The prevention of hyperacute rejection is the subject of European patent0495852 (Imutran). To make tissues more suitable for xenografting thispatent teaches that they should be associated with homologous complementrestriction factors, which prevent the complete activation of complementin the xenogeneic organ recipient

This approach has been developed and applied in order to producetransgenic animals with organs designed to survive hyperacute rejection(Squinto, 1996). Transgenic mice expressing human CD59, a complementregulator, on cardiac ECs have been produced (Diamond, 1995). The humanCD59 retained biological activity and complement was inhibited whentransgenic hearts were perfused with human plasma.

Transgenic pigs expressing human DAF and/or CD59 have been reported(McCurry, 1996). Cardiac rejection took twice as long to occur with thetransgenic xenografts than with controls.

Inhibiting delayed vascular rejection has not received the sameattention, although inhibitors of the coagulation cascade are well knownin the art and many have been well characterised.

For instance, tissue factor pathway inhibitor (TFPI) is known to inhibitthe function of the active complex which is normally formed betweentissue factor, factor VIIa, and factor Xa TFPI is a 276 residue solublepolypeptide whose positively charged C-terminus binds to heparinsulphate in the proteoglycan layer of ECs. It has been notionallydivided into three “Kunitz” domains: Kunitz domain I is responsible forbinding tissue factor and factor VIIa; domain II binds factor Xa; butthe functions of domain III are less clear (Hamamoto, 1993).

Tick anticoagulant peptide (TAP) is a specific and potent inhibitor offactor Xa. This 60 amino acid polypeptide has been purified from thesoft tick Ornithodoros moubata.

Many snake venoms also contain anticoagulant polypeptides. For instance,a 231 amino acid protein C activator has been purified from the venom ofthe snake Agkistrodon contortrix contortrix (McMullen, 1989; Kisiel,1987).

Hirudin is the anticoagulant protein utilised by the leech Hirudomedicinalis when extracting blood from its victim. It is highly potentand binds to thrombin at a 1:1 ratio with a dissociation constant in thefemtomolar range. The active site of thrombin is masked in the stablecomplex and so the hirudin prevents fibrinogen breakdown, thusinhibiting clot formation.

One possible approach for localising anticoagulants to the site ofrejection is to link hirudin to antibodies against E-selectin, which isexpressed on the surface of ECs during cell activation. This approachhas been shown to be effective in inhibiting clot formation in vitro(Kiely, 1995). Other possible strategies were recently reviewed by Bachet. al (1996).

P-selectin (also known as CD62) is also expressed on the surface of ECsduring cell activation. During synthesis it is targeted to secretorystorage granules in platelets and endothelial cells by sequencesresiding in its cytoplasmic domain (Disdier, 1992). In response to cellagonists, such as thrombin, the granules are rapidly redistributed andP-selectin is expressed on the cell surface (Green, 1994).

It is an object of the present invention to provide membrane-boundanticoagulant proteins. These proteins are suitable for inhibiting theclotting cascade at the surface of ECs, thus inhibiting in vivomechanisms responsible for organ rejection.

It is a further object to provide regulated expression of such moleculeson the surface of ECs such that coagulation inhibition occurs locallyduring conditions of organ rejection. The rejection might be xenogeneicor allogeneic.

It is yet a further object of the invention to provide biological tissuesuitable for transplantation, particularly for xenotransplantation.

DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is provided aprotein comprising a region with anticoagulant activity and a regionwhich can anchor said protein to a cell membrane. Preferably this is achimeric protein, that is to say the anchor region and anticoagulantregion are derived from different proteins.

The anticoagulant region can comprise the sequence of any anticoagulantpolypeptide. Examples of such anticoagulant polypeptides includeheparin, TAPs, antithrombin, hirudins, and TFPIs, along with theirfunctional derivatives, such as fragments and derivatives which retainanticoagulant activity. Anticoagulant derivatives of thrombin, normallya procoagulant, have also been reported (Dang, 1997).

Preferably the anticoagulant region comprises the sequence of a hirudin.Hirudins include hirudin, hirudin derivatives, analogs (“hirulogs”), andvariants (eg. hirudisins). For instance, it has been reported thatsulphation at Tyr-64 increases the anticoagulant activity of hirudin,and that hirudisin-2 is a more potent inhibitor of thrombin activitythan hirudin itself (eg. Knapp, 1992; Skern, 1990).

As an alternative, the anticoagulant region might comprise the sequenceof a tissue factor pathway inhibitor (TFPI). TFPIs include TFPI itselfand derivatives or analogs thereof which retain inhibitory activity.Preferably the TFPI sequence comprises Kunitz domains I and II of TFPIitself.

As a further alternative, the anticoagulant region might comprise thesequence of a tick anticoagulant peptide (TAP). TAPs include TAP itselfand derivatives or analogs thereof which retain inhibitory activity. Forinstance, the potency of FXa inhibition by TAP has been enhanced bysite-directed mutagenesis (eg. Mao, 1995).

Further alternative anticoagulant regions could, for instance, comprisethe sequence of a protein C activator, such as those isolated from snakevenom (eg. McMullen, 1989; Kisiel, 1987), or the sequence ofanticoagulants isolated from snake venoms which act other than viaprotein C activation, or their derivatives or analogs which retainanticoagulant activity.

The anchor region can be any entity which can attach the protein to acell membrane. Suitable examples include transmembrane sequences frommembrane proteins and GPI anchors. Preferably the anchor region is asequence capable of attaching the protein to a lipid bilayer, such asthe transmembrane regions of the HLA class 1 or CD4 proteins. It mayalso be desirable for the protein to comprise the cytoplasmic domainwhich is usually associated with said transmembrane regions, such as theCD4 cytoplasmic domain, and/or the extracellular domains immediatelyjuxtaposed with the cell membrane, such as CD4 domains 3 and 4.Alternatively the anchor region might be a sequence conferring on theprotein the ability to associate extracellularly with a membrane proteinwithout the protein itself being inserted into the cell membrane.

According to a second aspect of the invention, there is provided aprotein according to the first aspect further comprising a targetingsequence which prevents the protein from being constitutively expressedat the cell surface.

Preferably the targeting sequence is a polypeptide sequence which cantarget a nascent polypeptide to a secretory granule, and more preferablythe secretory granule is one which does not fuse with the cell's plasmamembrane until the cell is suitably stimulated. For example,Weibel-Palade bodies do not fuse with the plasma membrane until theendothelial cell surface is stimulated by a secretagogue, such asthrombin or fibrin (Wagner, 1993). Preferably the secretory granulefuses with the plasma membrane during EC activation which occurs duringorgan rejection.

Thus the targeting sequence is preferably one which targets a nascentpolypeptide to a Weibel-Palade body, such as the relevant sequence fromP-selectin. Most preferably the protein according to the second aspectof the invention comprises an anticoagulant sequence and thetransmembrane and cytoplasmic domains of P-selectin. The domains fromP-selectin thus provide both the anchor sequence and the targetingsequence.

According to a third aspect of the invention, there is provided apolynucleotide encoding a protein according to the present invention.Preferably the polynucleotide is DNA.

Preferably the polynucleotide comprises sequences suitable for theregulation of expression of protein according to the invention. Thisexpression can preferably be controlled, such as cell-specific control,inducible control, or temporal control. For instance, expression mightbe specific for ECs, or might be regulated in response to cellactivation.

According to a fourth aspect of the invention, there is provided avector comprising a polynucleotide according to the third aspect.

The term “vector” signifies a molecule which is capable of transferringa polynucleotide to a host cell. Preferably the vector is a DNA vectorand, more preferably, is capable of expressing RNA encoding a proteinaccording to the invention. Numerous suitable vectors are known in theart.

Preferably the vector is suitable for the production of a transgenicanimal. Vectors suitable for the generation of transgenic pigs, forexample, are described in Heckl-Östreicher (1995), McCurry (1996), White(1995), Yannoutsos (1995), and Langford (1996). Minigene vectorssuitable for the generation of transgenic mice are described in Diamond(1995).

According to a fifth aspect of the invention, there is provided adelivery system comprising a molecule of the first, second, third, orfourth aspects and means to deliver said molecule to a target cell.

Certain vectors according to the fourth aspect may also function assuitable delivery systems. Likewise, certain delivery systems accordingto this fifth aspect may also inherently be vectors, but this is notalways the case. For instance, a viral vector can also function as adelivery system, whereas a liposomal delivery system is not a vector.

The delivery system may be viral or non-viral. Non-viral systems, suchas liposomes, avoid some of the difficulties associated with virus-basedsystems, such as the expense of scaled production, poor persistence ofexpression, and concerns about safety. Preferably the delivery system issuitable for use in gene therapy. Numerous appropriate delivery systemsare known in the art.

Preferably, the delivery system will be targeted so that moleculesaccording to the present invention are taken up by cells suitable fortransplantation, or cells which have been transplanted. More preferablythe delivery system will be specific for these cells. For example, thedelivery system may be targeted to a specific organ, such as the heartor the kidney, or to a specific cell type, such as endothelial cells.

To achieve this the delivery system may, for example, be areceptor-mediated delivery system, being targeted to receptors found ontarget cells. For example, the delivery system may be targeted toreceptors found on heart cells, preferably to receptors foundexclusively on heart cells, or it may be targeted to receptors found onendothelial cells, preferably to receptors found exclusively onendothelial cells, or to receptors found on activated endothelial cells,such as E-selectin or P-selectin.

The delivery system is preferably suitable for the generation oftransgenic animals. For example, the delivery system may be targeted toa gamete, a zygote, or an embryonic stem cell.

According to a sixth aspect of the invention, there is provided a methodof transfecting a cell with a vector according to the invention. Thismay involve the use of a delivery system according to the invention.

The cell type is not restricted and may be prokaryotic or eukaryotic.Transfection can occur in vivo or ex vivo.

Where the cell is for use in transplantation, the cell is preferablyeukaryotic, more preferably an endothelial cell. The stable transfectionof porcine endothelial cells, for example, is described inHeckl-Östreicher (1995).

Preferably, the cell is suitable for the generation of a transgenicanimal. More preferably the cell is a gamete, a zygote, or an embryonicstem cell. The transfection of murine ova by microinjection to generatetransgenic mice, for example, is described in Diamond (1995), and themicroinjection of porcine zygotes, for instance, to generate transgenicpigs is described in Yannoutsos (1995), Langford (1996), and White(1995).

According to a seventh aspect of the invention, there is provided a celltransfected according to the sixth aspect.

To increase the efficacy of inhibition of the coagulation cascade, thecell is preferably able to express two or more different proteinsaccording to the invention, each of which inhibits the coagulationcascade at a different stage. For example, the anticoagulant region inone protein might comprise a TFPI, whilst in the other it comprises ahirudin.

According to an eighth aspect of the invention, there is providedbiological tissue comprising a cell according to the invention. The term“biological tissue” as used herein includes collections of cells,tissues, and organs. Accordingly the definition includes, for example,fibroblasts, a cornea, nervous tissue, a heart, a liver, or a kidney.

According to a ninth aspect of the invention, there is provided ananimal comprising a cell and/or biological tissue according to theinvention. Preferably the animal is suitable for the production oforgans for transplantation into humans. Preferably the animal is amammal, and more preferably it is a transgenic pig or a transgenicsheep.

The animal might be treated whilst alive such that it comprisestransgenic biological tissue (ie. treated by gene therapy). Preferably,a live animal is transfected with a vector according to the invention inorder to produce a transgenic animal. For example, a vector according tothe invention could be specifically delivered to endothelial cells in apig to produce transgenic organs suitable for xenotransplantation.

Alternatively, the animal might be born as a transgenic animal. Varioussuitable approaches for generating such transgenic animals are known inthe art (eg. Bradley & Liu, 1996; Clarke, 1996; Wheeler, 1994). Forexample, direct manipulation of the zygote or early embryo, bymicroinjection of DNA for instance, is well known, as is the in vitromanipulation of pluripotent cells such as embryonic stem cells.Retroviral infection of early embryos has proved successful in a rangeof species, and adenoviral infection of zona-free eggs has beenreported. Transgenesis and cloning of sheep by nuclear transfer has alsobeen described (eg. WO97/07668).

According to a tenth aspect of the invention, there is provided a methodof rendering biological tissue suitable for transplantation, comprisingexpressing one or more proteins according to the present invention insaid biological tissue, preferably in its endothelial cells. Thebiological tissue may be so rendered either in vivo or ex vivo. Forexample, an animal organ may be in vivo transfected with a vectoraccording to the invention, or an organ could be transfected ex vivobefore transplantation or in vivo after transplantation.

According to an eleventh aspect of the invention, there is provided amethod of transplantation comprising transplanting biological tissueaccording to the invention from a donor animal into a recipient animal.Preferably the method is for xenotransplantation and the donorbiological tissue is xenogeneic with respect to the recipient animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows maps of hirudin-CD4 chimeric proteins and constructsaccording to the invention. (A) HLA-hirudin-CD4 constructs with glycinelinkers. (B) HLA-hirudin-CD4 construct with human P-selectin C-terminal,with the specific targeting sequence underlined. Transmembrane (TM),stop transfer (ST), and cytoplasmic (C) regions of CD4 are indicated.

FIG. 2 shows FACS profiles for HLA-hirudin-CD4 constructs expressed inDAP.3 fibroblasts.

FIG. 3 shows FACS profiles for HLA-hirudin-CD4-P-selectin cDNAconstructs expressed in CHO-K1.

FIG. 4 shows that hirudin-CD4 expressing fibroblasts bind thrombin.

FIG. 5 shows the specificity of thrombin binding to cells expressinghirudin-CD4.

FIG. 6 shows thrombin binding to CHO-K1 cells transfected withHLA-hirudin constructs.

FIG. 7 shows that inactivation of thrombin abolishes thrombin binding tohirudin-CD4 at the cell surface. Cells expressing hirudin-G2-CD4 wereincubated with thrombin or inactivated thrombin and stained for thrombinbinding with anti-prothrombin or anti-thrombin-hirudin antibodies.

FIG. 8 shows maps of TFPI-CD4 chimeric proteins and constructs accordingto the invention.

FIG. 9 shows flow cytometry profiles of DAP.3 cells expressing TFPItethered to the cell surface.

FIG. 10 shows specific FXa binding to cell surface bound TFPI₁₋₂₇₆-CD4and TFPI₁₋₁₈₃-CD4.

FIG. 11 shows the blocking of FXa binding by a polyclonal anti-TFPIimmunoglobulin fraction.

FIG. 12 shows the blocking of FXa binding by monoclonal antibodiesdirected against Kunitz domains I and II.

FIG. 13 shows the inhibition of FXa by cells expressing TFPI₁₋₂₇₆-CD4and TFPI₁₋₁₈₃-CD4. The mean time for a FXa-specific chromogenicsubstrate to reach OD₄₀₅=0.1 is shown for transfected DAP.3 cellsincubated with FXa. Values for control cells were subtracted and errorbars indicate standard deviations.

FIG. 14 shows that an active TF₁₋₂₁₉/FVIIa complex is required formaximal binding to TFPI-CD4 chimeric proteins.

FIG. 15 shows the specificity of thrombin binding to immortalisedporcine endothelial cells (IPEC) expressing hirudin-CD4, and also showsthe effect of cell-surface hirudin-CD4 expression on clotting times.

FIG. 16 shows the distribution of ACTH and hirudin in D16/16 cells, asrevealed by fluorescence.

FIG. 17 shows the change in cellular distribution ofhirudin-CD4-P-selectin after PMA stimulation

FIG. 18 shows that TFPI-CD4 expressed on IPEC retains its bindingproperties.

FIG. 19 shows the competitive binding of porcine and human tissuefactors.

FIG. 20 shows that TFPI-CD4 prolongs clotting times when expressed onIPEC surface.

FIG. 21 shows the anti-coagulant effect of co-expression of TFPI-CD4 andhirudin-CD4.

DESCRIPTION OF EMBODIMENTS

1. Hirudin Fused with HLA Class I Signal Peptide and Linked to Domains 3and 4 of Human CD4 is Tethered to the Cell Membrane

To express heterologous hirudin constructs in mammalian cells, the cDNAfor the membrane-targeting signal peptide leader sequence from human HLAclass 1 A2.1, amino acids −1 to −24 (Holmes, 1987), was fused to hirudinvariant 1 (Dodt, 1984) using PCR with overlapping extension (FIG. 1).

The HLA A2.1 leader sequence was amplified using primers: <SEQ ID 1>5′-cagtgtcgacggatccatggccgtcatggcgccccga-3′ [hla-1] (introducing SalIand BamHI restriction sites) and: <SEQ ID 2>5′-gtcagtgtaaacaaccgcccaggtctgggtcagg-3′

The hirudin sequence was amplified using primers: <SEQ ID 3>5′-acccagacctgggcggttgtttacactgactgcacc-3′ and <SEQ ID 4>5′-gacgctgcagaattcttgcaggtattcttccgggatt-3′ [hir-3](introducing distal EcoRI and PstI sites).

The resulting PCR products (108 and 228 bp) were purified by agarose gelelectrophoresis and then used in a third PCR using flanking primershla-1 and hir-3. The resulting PCR product (300 bp) was digested withSalI and BamHI and subcloned into pBluescript SK(+) (Stratagene).

An anchor consisting of a cDNA encoding for CD4 domains 3 and 4 (Maddon,1985) in conjunction with the stop transfer sequence (ST), transmembraneand cytoplasmic domains of CD4 (CD4₁₆₆₋₄₃₅) was added to the HLA-hirudincassette.

To ensure that hirudin stayed mobile and active when linked by itsC-terminal to the CD4 anchor, however, 3 different glycine linkerlengths were made (designated G1 to G3—FIG. 1A):

for glycine linker 1 (G1; GGSGG), the oligonucleotide pair consisted of5′-aattaggaggttctggaggctgca-3′ <SEQ ID 5> (containing a mutated EcoRIrecognition sequence and a PstI site) and 5′-gcctccagaacctcct-3′; <SEQID 6>

-   -   glycine linkers 2 (G2) and 3 (G3) consisted of the core sequence        (GGSGG) repeated two or three times, respectively.

These linkers were introduced into the 3′ end of the HLA-hirudinfragment.

The glycine linker oligonucleotides were annealed and each ligated intothe EcoRI/PstI site of plasmids containing the HLA-hirudin cassette,prior to the insertion of the CD4 anchor.

CD4₁₆₆₋₄₃₅ was amplified using primers:5′-tgtctgcaggaaccagaagaaggtggaattca-3′ <SEQ ID 7> (introducing PstI andEcoRI sites) and: 5′-gtgggatccgcctggcctcgtgcctcaa-3′ <SEQ ID 8>(containing a distal BamHI).

The resulting PCR product was cloned into pBluescript and sequenced. InCD4₁₆₆₋₄₃₅, V³²⁸ was found to be mutated to A³²⁸. The PstI/BamHI CD4fragment was subcloned into HLA-hirudin-G1, -G2, & -G3 plasmids, andthese constructs were verified by DNA sequence analysis.

Each of the three cDNA constructs were subcloned into the BamHI site ofthe mammalian expression vector pHβActpr-1gpt (Gunning, 1987),containing the human β-actin enhancer and promoter region in conjunctionwith an SV40 enhancer element driving the gpt resistance gene, allowingthe selection of clones in the presence of mycophenolic acid (FIGS. 1C &1D). The orientation of the final constructs was verified by restrictionendonuclease mapping.

Vectors containing the individual HLA-hirudin-G1/2/3-CD4 constructs weretransfected into mouse fibroblast cell line DAP.3 (Marguelies, 1983)with calcium-phosphate according to standard protocols. After 18 hoursgrowth in DMEM medium (Gibco) supplemented with 5% fetal calf serum,ampicillin, streptomycin, and glutamine, cells were glycerol treated for30 seconds. Cells were then washed twice with phosphate buffered saline(PBS), and new medium including xanthine, hypoxanthine, and mycophenolicacid to a final concentration 12 μg/ml, was added.

For a negative control, DAP.3 cells transfected with a human class IIconstruct expressing HLA-DR (cell line 531) (Lechier, 1988) grown inidentical mycophenolic acid-containing culture medium.

Surviving clones were tested for hirudin and CD4 expression by FACSusing murine monoclonal antibodies 4158-81-7 (Schlaeppi, 1991) and OKT-4(Reinherz, 1979) respectively. 10⁵ cells were stained with the murineantibodies for 30 minutes on ice and a FITC-conjugated sheep anti-mousepolyclonal antibody was added as a secondary layer.

As shown in FIG. 2, these hirudin-CD4 constructs were well expressed atthe cell surface of DAP.3. No significant difference in expressionlevels was detected between hirudin-CD4 with the three different glycinelinker lengths.

Therefore anticoagulant polypeptides can be stably expressed on the cellsurface.

2. Hirudin-CD4 with a Targeting Sequence from the C-terminal ofP-selectin is Expressed at the Cell Surface of CHO-K1

In addition to the HLA-hirudin-G1/2/3-CD4 constructs, two moreconstructs were synthesised with targeting sequences derived from humanP-selectin (FIG. 1B). The transmembrane region from CD4 was used forthese constructs, while the stop transfer sequence and C-terminal werereplaced with the corresponding sequences from P-selectin (Johnston,1989).

To fuse CD4 domains 3 and 4 plus the transmembrane region (CD-4₁₆₆₋₃₉₅)with the stop transfer sequence and cytoplasmic regions 1 and 2 of humanP-selectin (P-sel₇₅₄₋₇₈₉) (McEver 1989), PCR with overlapping extensionwas performed. For amplification of the CD4 part of the molecule,primers: 5′-tgtctgcaggaaccagaagaaggtggaattca-3′ [CD4-5] <SEQ ID 7>(introducing PstI and EcoRI restriction sites) and:5′-gtctgaaacgctttctgaagaagatgcctagcccaatgaaaagcaggaggccg-3′ <SEQ ID 9>

were used. In parallel, to amplify the C-terminal region of P-selectin,primers: <SEQ ID 10> 5′-tgggctaggcatcttcttcagaaagcgtttcagacaaaaaga-3′and <SEQ ID 11> 5′-gaccaggatccggacaggtctctta-3′ [P-selN3](introducing a distal BamHI site) were used.

After purification of resulting PCR products from agarose gels, a thirdPCR was run using the flanking primers CD4-5 and P-selN3. The resultingPCR product (832 bp) was digested with PstI and BamHI, subcloned intopBluescript, and sequenced. Thereafter, the CD4-P-sel fragment(CD4₁₆₆₋₃₉₅-P-sel₇₅₄₋₇₈₉) was excised with PstI/BamHI and subcloned intoplasmids containing HLA-hirudin-G1 or -G2.

The final HLA-hirudin-G1/G2-CD4-P-selectin constructs were subclonedinto the BamHI site of pHβActpr-1gpt and transfected into CHO-K1 cells(ATCC CCL61), grown in RPMI 1640 medium (Gibco) supplemented with 5%fetal calf serum, anpicillin, streptomycin, and glutamine.

Transfection was by electroporation according to standard protocols.Briefly, 5×10⁶ cells were resuspended in 350 μl serum-free medium andtransferred to a 1 ml electroporation cuvette with a 0.4 cm spacebetween electrodes (Bio-Rad). After addition of 10 μg plasmid DNA in 150μl, samples were gently shaken and kept on ice. Cells were subjected toelectroporation at infinite resistance, 960 μF and 350 V in a GenePulser apparatus (Bio-Rad). The day after electroporation, cells werewashed twice with PBS and new medium including mycophenolic acid,xanthine, and hypoxanthine was added.

Recently it was shown that when CHO-K1 cells were transfected withP-selectin cDNA, P-selectin protein was not accumulated intracellularly,but was expressed at the cell surface (Disdier, 1992). In the CHO-K1transfectants produced above, both hirudin-G1-CD4-P-selectin andhirudin-G2-CD4-P-selectin were expressed at the surface as judged bystaining with OKT-4 and 4158-81-7 monoclonals (FIG. 3). The negativecontrol used was a CHO-K1 cell line expressing TFPI fused to CD4 domains3 and 4 (TFPI-CD4₁₆₆₋₄₃₅), grown in the same mycophenolicacid-containing medium.

As a positive control, CHO-K1 cells were transfected with full lengthhuman P-selectin (Johnston, 1989), which was subcloned as a 3142 bp SalIfragment into pHβActpr-1neo containing an SV40-driven neomycin (G418)resistance gene. These cells were treated with 400 μg/ml G418 and after2 weeks individual clones were picked with cotton swabs and transferredto 12-well plates. Surviving clones were analysed for hirudin and CD4expression using 4158-81-7 at 10 μg/ml and an undiluted OKT-4 hybridomasupernatant.

Human P-selectin was detected by anti-CD62 mAb (Becton Dickinson),according to the manufacturer's recommendations. A similar FACS profileas with hirudin-CD4-P-selectin was observed for these CD62-labeled cells(FIG. 3E), confirming that CHO-K1 cells express P-selectin at the plasmamembrane.

Thus, chimeric proteins comprising the P-selectin targeting sequenceremain functional when expressed at the cell surface.

3. Hirudin Anchored to the Cell Surface Binds Thrombin as Detected withSpecific Antibodies

To test whether hirudin tethered in this way to the cell surface retainsits thrombin binding activity, the following binding assay was used.

Stably transfected cells were grown in T75 culture flasks for 36 hoursbefore each experiment. DAP.3 cells were detached using a cell scraper,whilst CHO-K1 cells were detached from the plastic by treatment withPBS, 5 mM EDTA for 10 minutes at 37° C. After 4 washes with PBScontaining 0.1% BSA (w/v), 2.5×10⁵ cells in 150 μl were incubated for Ihour at 37° C. with increasing concentrations of thrombin. The cellswere washed four times with PBS containing 0.1% BSA and furtherincubated for 30 minutes on ice with rabbit anti-human prothrombinimmunoglobulins (10 μg/ml in 100 μl) (Dakopatts). After two furtherwashes, cells were incubated for 30 minutes with FITC-conjugated swineanti-rabbit immunoglobulins (Dakopatts). Finally, transfectants werewashed three times and analysed by flow cytometry.

As shown in FIG. 4, hirudin expressed at the cell surface retains theability to bind thrombin and glycine linker length did not influencethrombin binding.

To assess the amount of thrombin needed to saturate the hirudin-CD4expressing cells, two clones were incubated with thrombin up to 82 U/ml.When percentage positive cells was analysed, transfectants weresaturated at 41 U/ml thrombin (FIG. 4C). According to the meanfluorescence intensities (mfi), however, cells were not saturated evenat 82 U/ml (FIG. 4D). At these high experimental thrombin concentrationsthe background binding to control cells expressing HLA-DR increasedsignificantly.

To elucidate the specificity of thrombin binding to hirudin-CD4 further,blocking experiments were carried out. DAP.3 HLA-hirudin-G3-CD4transfectants were pre-incubated on ice for 30 minutes with 10 μg/mlanti-hirudin mAb or appropriate controls (mouse IgG1 and IgG2a,Dakopatts) for 30 minutes on ice, and washed twice in PBS containing0.1% BSA before incubating with thrombin for 1 hour at 37° C. as above.Thrombin binding was analysed as above.

Pre-incubation with 4158-81-7 inhibited specific thrombin binding tohirudin-CD4 (FIG. 5A). Thrombin binding by hirudin-CD4 was demonstratedby incubation with thrombin and comparing labelling with mAb 4107-76-1(Schlaeppi, 1991) and anti-prothrombin immunoglobulins. 4107-76-1 isdirected against the hirudin-thrombin complex and detects neitherhirudin without thrombin nor thrombin bound to endogenous thrombinreceptors. As shown in FIG. 5B, thrombin binding detected with 4107-76-1paralleled the binding observed with the anti-prothrombin immunoglobulinfraction.

Thus hirudin expressed on the surface of DAP.3 cells retains specificthrombin binding.

Immortalised porcine epithelial cells (IPEC) were transfected withhirudin-CD4 in the same way. As shown in FIG. 15A, only the transfectedcells bound thrombin, and this was blocked by the 4158-81-7 in adose-dependent manner (FIG. 15B). A human plasma recalcification testsystem was used for further investigation of the functional effect ofexpressing surface-tethered hirudin on IPEC. As shown in FIG. 15C,untransfected IPEC shortened the clotting time of recalcified plasma toapproximately 170 seconds, compared with a control clotting time 370 sin the absence of cells. Preincubation with IL-1, which induces TFexpression, further reduced the clotting time to below 100 s. Incontrast, clotting times for transfected IPEC were prolonged, even afterpreincubation with IL-1-induced TF expression. Incubation with 4158-81-7reduced the anticoagulant effect in a dose-dependent manner, indicatingthat the effect was due to the presence of cell-surface hirudin (FIG.15D).

Hirudin expressed on the surface of IPEC thus binds thrombin and alsoinhibits the clotting of human plasma.

4. Hirudin-CD4-P-selectin Expressed by CHO-K1 Cells Binds Thrombin

To investigate whether hirudin-CD4-P-selectin also binds thrombin whenexpressed at the surface of CHO-K1 cells, these cells were incubatedwith thrombin for 1 hour at 37° C. After staining with anti-prothrombinimmunoglobulins and addition of a second FITC-labelled antibody layer,cells were analysed by flow cytometry.

A distinct binding profile was detected, as shown in FIG. 6A. Withanti-prothrombin immunoglobulins, background thrombin binding to CHO-K1cells expressing an irrelevant protein linked to CD4 was detectableafter incubation with fairly low concentrations of thrombin. However,specific thrombin binding to hirudin was verified by staining with thespecific anti-hirudin/thrombin mAb 4107-76-1 (FIG. 6B). With thisantibody, background binding by the control CHO-K1 cells wasundetectable. It is also evident from FIG. 6 that clones expressinghirudin appeared to bind thrombin non-specifically to a different degreeimplying that they had different expression levels of endogenousthrombin receptors. This variation in non-specific binding was confirmedwith several other clones.

For comparison, results from two CHO-K1 transfectants expressinghirudin-G1-CD4 and hirudin-G2-CD4 (ie. no P-selectin sequence) are shownin FIGS. 6C and 6D. Except for a slightly increased thrombin binding dueto better expressed chimeric proteins (higher mfi's), no majordifferences in binding profiles were detected compared to transfectantsexpressing hirudin linked to the CD4-P-selectin anchor.

5. Hirudin-CD4-P-selectin is Stored in Secretory Granules and can beReleased on Activation

To examine intracellular accumulation of hirudin and its route fromsecretory granules to the cell surface, a secretory murine pituitarycell line (D16/16) was transiently transfected with cDNA encoding eitherhirudin-CD4-P-selectin or hirudin-CD4. This cell line was chosen for tworeasons. Firstly, these cells are known to express ACTH in specificstorage granules, which are discharged at the cell surface on activationwith phorbol esters. Secondly, endothelial cells (which would appear tobe the ideal cell type to investigate intracellular targeting of theP-selectin construct) rapidly lose their Weibel-Palade storage granulesduring in vitro culture.

48 hours after transfection, D16/16 cells were stained with antibodiesagainst hirudin and ACTH. In cells transfected withhirudin-CD4-P-selectin, hirudin was detected in granules evenlydistributed in the cytoplasm (FIG. 16A). The same pattern of granuledistribution was seen with ACTH-specific staining, implyingco-localisation with hirudin (FIG. 16B). This was verified when bothantibodies were used for staining (FIG. 16C).

In contrast, D16/16 cells transfected with hirudin-CD4 did notaccumulate hirudin in intracellular granules, but expressed high levelsof hirudin at the cell surface (FIG. 16D). Dual staining (FIG. 16F)revealed only slight co-localisation of hirudin and ACTH.

Cells expressing hirudin-CD4-P-selectin were activated with phorbolester PMA, and were analysed by flow cytometry. 4158-81-7 did not detectany hirudin at the cell-surface in unstimulated cells (FIG. 17A). After30 minutes of PMA-stimulation, however, hirudin was detected at thecell-surface (FIG. 17B). Furthermore, activated D16/16 cellsspecifically bound to thrombin, unlike non-activated cells (FIG.17C—stained with 4107-76-1).

Thus, by using the granule-containing pituitary cell line D16/16, it wasclearly demonstrated that hirudin-CD4-P-selectin can be targeted tospecific intracellular storage granules, and that functional chimericmolecules can be released and exposed at the cell surface uponactivation.

6. The Interaction Between Thrombin and Hirudin-CD4 is Abolished whenthe Catalytic Site of Thrombin is Inactivated.

Specific thrombin binding to hirudin-CD4 with and without P-selectintargeting sequence was clear (FIGS. 4 and 6). To strengthen thespecificity of the thrombin-hirudin interaction further, thrombin (210nmol in 50 μl Tris-buffered saline (TBS), 0.1% BSA, pH 7.4) waspre-incubated for 1 hour at 37° C. with either:

-   -   native fill-length hirudin (Biopharm) at a 10-fold molar excess;    -   D-Phe-Pro-Arg chloromethyl ketone dihydrochloride (“PPACK-HCl”)        (Calbiochem) at 100-fold molar excess; or    -   a synthetic C-terminal hirudin dodecapeptide analog comprising        hirudin residues 53-64, with sulfato-Tyr64 (American        Diagnostica) at 100-fold molar excess.

The thrombin-dependent catalytic activity was analysed with a smallchromogenic oligopeptide substrate (H-D-Phe-Pip-Arg-pNA2HCl (“S-2238”)(Quadratech).

To ascertain whether thrombin was inactivated by PPACK-HCl and hirudin,5 μl of each reaction mixture were diluted with 95 ml TBS, 0.1% BSA andincubated with 50 μl 4 mM S-2238 for 10 minutes at 37° C.

As expected, no chromogenic conversion was observed with thrombinincubated with PPACK-HCl or hirudin as compared to thrombin incubatedwithout inhibitor, whereas the dodecapeptide did not influencethrombin-dependent catalytic activity as measured by cleavage of S-2238.

The three different preparations were added to transfectants expressinghirudin tethered to the cell surface. Using the procedure describedabove, thrombin binding was investigated with the anti-prothrombin oranti-hirudin-thrombin antibodies. As can be seen in FIG. 7A, thrombininactivated with hirudin or PPACK-HCl was not bound by hirudin expressedat the cell surface of DAP.3. In addition, only a partialthrombin-dodecapeptide complex binding was observed. In contrast toDAP.3 transfectants, CHO-K1 cells displayed a relatively highthrombin-PPACK-HCl binding (FIG. 7B). This interaction was found to beunspecific as illustrated with the anti-hirudin-thrombin mAb 4107-76-1.No specific thrombin-PPACK-HCl-hirudin binding was detected.

This confirms that hirudin tethered to the cell surface specifically andstrongly binds thrombin at its catalytic site.

7. Full Length and Truncated TFPI Anchored to CD4 Domains is Expressedat the Cell Surface

In order to tether TFPI to the cell membrane, a fusion proteinconsisting of human CD4₁₆₆₋₄₃₅ linked either to full length TFPIincluding all three Kunitz domains (TFPI₁₋₂₇₆) or to a truncated form ofTFPI lacking Kunitz domain III and the C-terminal (TFPI₁₋₁₈₃) (Wun,1988) (FIG. 8). These were synthesized in a similar way to thatdescribed above for hirudin, with the TFPI and CD4 sequences being fusedusing a cassette cloning strategy, but unlike hirudin, TFPI is amammalian protein and hence contains an endogenous signal peptide.

DNA encoding the N-terminal portion of TFPI including Kunitz domains Iand II (675 bp) was amplified using the primers:5′-catcgtcgacggatcctagatgatttacacaatgaagaaagtacatgcactttgggc-3′ <SEQ ID12> (introducing SalI and RamHI restriction sites); and5′-ggacctgcagaattcaaaaaggctgg-3′ <SEQ ID 13> (containing EcoRI and PstIsites).

DNA encoding the third Kunitz domain together with the C-terminal end ofTFPI (315 bp) was amplified using primers:5′-agcctttttgaattccacggtccctcat-3′ <SEQ ID 14> (with an EcoRI site); and5′-cattgctataacaactgcagatatttttaac-3′ <SEQ ID 15> (containing a PstIsite).CD4₁₆₆₋₄₃₅ was amplified as described above.

By the introduction of restriction sites into the 3′ end of theTFPI₁₋₁₈₃ cDNA and the 5′ end of the TFPI₁₈₄₋₂₇₆ cDNA, H¹⁸⁴ and G¹⁸⁵were mutated to C¹⁸⁴ and R¹⁸⁵ in the recombinant fusion proteins (FIG.8). Furthermore, P¹⁸⁶ was mutated to S⁸⁶. The stop codon of TFPI wasremoved by introducing a PstI site, thus mutating M²⁷⁶ to I²⁷⁶, and theaddition of amino acid C²⁷⁷. In the course of introducing a PstI site inthe N-terminal end of CD4 domain 3, L¹⁶⁴ and Q¹⁶⁵ were mutated to C¹⁶⁴and R¹⁶⁵, respectively. In the TFPI₁₈₄₋₂₇₆ cDNA, K²⁶⁵ was found to bemutated to E²⁶¹ and in CD4₁₆₆₋₄₃₅ V³²⁸ was mutated to A³²⁸ (as describedabove).

All PCR products were cloned into pBluescript SK(+).

The complete TFPI-CD4 cDNAs were ligated into the BamHI site of thepHβActpr-1gpt expression vector.

As above, DAP.3 cells, maintained in supplemented DMEM were transfectedwith calcium-phosphate as above. Clones were analysed for TFPI and CD4expression by FACS using murine anti-human TFPI mAbs 4903 or 4904(American Diagnostica), both at 10 μg/ml, and an undiluted OKT-4hybridoma supernatant (Reinherz, 1979). 4903 is directed against Kunitzdomain I, whereas 4904 is directed against Kunitz domain II. 10⁵ cellsfor each sample were analysed and, as above, cell line 531 was used as acontrol.

As shown in FIG. 9, both TFPI₁₋₂₇₆-CD4 and TFPI₁₋₁₈₃-CD4 can beexpressed at the cell surface.

8. TFPI₁₋₁₈₃-CD4 and TFPI₁₋₂₇₆-CD4 Tethered to the Cell Surface ConferFXa Binding

To test whether TFPI tethered in this way to the cell surface retainsits FXa binding activity, the following binding assay was used.

Stably transfected DAP.3 cells were detached by treatment with PBS, 5 mMEDTA for 10 minutes at 37° C. After 4 washes with excess PBS, 0.1% BSA(w/v), 2.5×10⁵ cells in 100 μl were incubated for 1 hour at 37° C. withincreasing concentrations of FXa.

Cells were then washed twice and further incubated for 30 minutes on icewith 10 μg/ml rabbit anti-human FXa immunoglobulins (RAFX-IG, EnzymeResearch Laboratories) in 100 μl. After two additional washes, cellswere incubated for 30 minutes with FITC-conjugated swine anti-rabbitpolyclonal immunoglobulins and analysed by flow cytometry.

As shown in FIG. 10, DAP.3 cells expressing TFPI₁₋₂₇₆-CD4 andTFPI₁₋₁₈₃-CD4 at the cell surface strongly bound FXa in a dose-dependentfashion (FIG. 10), with significant binding detected at 0.02 nM. Nodifference in FXa binding was detected between full length and truncatedTFPI-CD4.

It was also possible to block FXa binding with a polyclonal anti-TFPIimmunoglobulin fraction (4901) or with monoclonals 4903 and 4904.

Cells were incubated on ice for 30 minutes with 4901, 4903, or 4904 atincreasing concentrations, using an anti-haemoglobin antiserum(Dakopatts) as a negative control. The cells were then washed twice inPBS, 0.1% BSA, and further incubated with 5 nM FXa for one hour at 37°C. The cells were then washed and incubated with RAFX-IG as above andanalysed for FXa binding by FACS.

FXa binding to TFPI₁₋₂₇₆-CD4 decreased 27% and 55% at 10 and 80 μg/mlpolyclonal 4901, respectively, compared with cells incubated with theirrelevant anti-haemoglobin polyclonal control (FIG. 11A). DiminishedFXa binding was also found for TFPI₁₋₁₈₃-CD4 cells pre-incubated with4901 (FIG. 11B).

When TFPI₁₋₂₇₆-CD4 was blocked with either 4903 or 4904, 33% less FXabinding was observed at 40 μg/ml mAb, compared with isotype-matchedmouse immunoglobulins (FIG. 12). No significant difference in blockingactivity was detected between mAbs 4903 and 4904.

This demonstrates for the first time that TFPI retains its FXa bindingactivity when expressed as a membrane-bound fusion protein.

9. TFPI₁₋₁₈₃-CD4 and TFPI₁₋₂₇₆-CD4 are Both Functionally Active AgainstFXa

To determine whether TFPI tethered to the cell surface retained itsability to inhibit the function of FXa, the proteolytic activity of FXawas analysed using the chromogenic substrateN-a-Z-D-Arg-Gly-Arg-pNA·2HCl (“S-2765”) (Quadratech).

Transfected DAP.3 cells were detached as described above and washed 4times with excess TBS, pH 7.4, 0.1% BSA. 0.5×10⁶ cells (in 100 [μl) perwell were incubated for 1 hour at 37° C. with various concentrations ofFXa. 50 μl of 4 mM S-2765 were added and cells were further incubatedfor 2 hours at 37° C. OD₄₀₅ was measured every 30 seconds and the timerequired to reach OD₄₀₅=0.1 was determined, showing remaining activeFXa.

FXa activity was inhibited by expressed TFPI-CD4 in a dose dependentmanner with the greatest inhibition noted when low concentrations of FXa(0.16 nM) were added (FIG. 13). In a series of experiments, nosignificant difference in FXa inhibition was observed between cellsexpressing TFPI₁₋₁₈₃-CD4 or TFPI₁₋₂₇₆-CD4.

Thus, Kunitz domain II retains its function when tethered to the cellsurface in both TFPI₁₋₁₈₃-CD4 and TFPI₁₋₂₇₆-CD4.

10. TF₁₋₂₁₉/FVIIa Complex Binds Irrespectively of the Presence of theThird Kunitz Domain

Binding of tissue factor and factor VIIa can be used to confirm whetherKunitz domain I also retains its function.

Recombinant human TF₁₋₂₁₉ and FVIIa were produced in E. coli and CHO-K1,respectively (O'Brien, 1994). These were mixed in equimolarconcentrations and incubated at 25° C. for 15 minutes to obtain aTF₁₋₂₁₉/FVIIa complex.

Polyclonal rabbit immunoglobulins against human TF were producedaccording to standard methods.

DAP.3 cells expressing either TFPI₁₋₂₇₆-CD4 or TFPI₁₋₁₈₃-CD4 wereincubated with 5 nM FXa for 1 hour at 37° C. Cells were washed twice andTF₁₋₂₁₉/FVIIa complex was added to 2.5×10⁵ cells in 100 μl. After 1 hourat 37° C. transfectants were washed twice and incubated with 50 μlpolyclonal rabbit anti-TF immunoglobulins (2.5 μg/ml) for 30 minutes onice followed by 2 washes, and further incubation with FITC-conjugatedswine anti-rabbit immunoglobulins. Positive cells were analysed by flowcytometry.

TF₁₋₂₁₉/FVIIa bound equally efficient to both TFPI₁₋₂₇₆-CD4 (FIG. 14A)and TFPI₁₋₁₈₃-CD4 (FIG. 14B), while no binding at all was detected tocontrol cell line 531.

To confirm specific binding to Kunitz domain I by the TF₁₋₂₁₉/FVIIacomplex, FVIIa was inactivated by pre-incubation with1,5-dansyl-Glu-Gly-Arg-chloromethyl ketone, dihydrochloride(“1,5-DNS-GGACK.HCl”). This binds to the active site of FVIIa andinhibits binding to TFPI whilst not affecting the formation of theTF₁₋₂₁₉/FVIIa complex (Bajaj, 1992).

FVIIa was first incubated with a 100-fold molar excess of1,5-DNS-GGACK.HCl for 18 hours at 20° C. and repurified by ion-exchangechromatography. Active-site inhibited FVIIa (FVlIai) was incubated withan equimolar concentration of TF₁₋₂₁₉ at 25° C. for 15 minutes and thenadded to 2.5×10⁵ cells in 100 μl. Subsequent steps were as describedabove.

As can be seen from FIG. 14, significantly less TF₁₋₂₁₉/FVIIai complexbound to TFPI-CD4 expressing cells as compared to bound “active”TF₁₋₂₁₉/FVIIa. No difference was observed between DAP.3 transfected withTFPI₁₋₂₇₆-CD4 or TFPI₁₋₁₈₃-CD4.

Thus Kunitz domain I also retains its function when tethered to the cellsurface in TFPI₁₋₁₈₃-CD4 and TFPI₁₋₂₇₆-CD4. It is therefore apparentthat TFPI tethered at the cell surface is functionally active as awhole.

11. TFPI-CD4 Expressed on IPEC Binds Relevant Human Clotting Factors andPorcine TF

As shown in FIG. 18, the TFPI-CD4 fusion protein can be expressed onIPEC and retains the ability to bind FXa and FVIIa. To demonstrate thatTFPI can physically interact with porcine TF, a competitive inhibitionapproach using soluble human TF was adopted. As shown in FIG. 19A, inthe presence of saturating concentrations of FXa and FVIIa, the bindingof soluble human TF to TFPI-transfected IPEC (pre-treated with IL-1α)was significantly reduced compared to the binding by TF-negative controltransfectants (not IL-1α activated). This suggests that porcine TF wascompeting with soluble human TF for VIIa, and therefore for TFPIbinding. FIG. 19B supports this, showing that binding of soluble humanTF to TFPI-CD4-transfected IPEC (IL-1α pre-activated) was increased ifthe transfectants were incubated with increasing concentrations ofantibody against porcine TF. The effect of this antibody could reflectinhibition of the interaction between porcine TF and FVIIa, or betweenporcine TF-VIIa complexes and TFPI-CD4. Either way, the results suggestthat the TFPI-CD4-fusion protein expressed on the surface of IPECphysically interacts with porcine TF-FVIIa.

12. TFPI-CD4 Expressed on IPEC Inhibits TF-dependent Fibrin Generation

FIG. 20A shows the results of a single representative experiment toillustrate the procoagulant phenotype of TFPI-CD4-transfected IPEC. Thepresence of the fusion protein on transfected cells consistentlyprolonged clotting times when compared with control IPEC. This effectwas only observed, however, after IL-1α activation—TFPI-CD4 expressionhad no influence on clotting times when TF-negative IPEC were used.Thus, the TFPI-CD4, as expected, inhibited TF-dependent, but notTF-independent fibrin generation. An anti-TFPI antibody, used inincreasing concentrations during a pre-incubation step, was able tonormalise clotting times back to those seen with untransfectedIL-1α-activated control IPEC (FIG. 20B), indicating that theprolongation of clotting times in the presence of the transfected cellswas due entirely to the specific inhibitory action of TFPI.

13. Expression of a Protein C Activator at the Cell Membrane

To express heterologous constructs comprising the protein C activatorisolated from the venom of Agkistrodon contortrix contortrix (McMullen,1989; Kisiel, 1987), a cDNA encoding the protein was synthesised. Theprotein sequence is <SEQ ID 16>: V I G G D E C N I N E H R F L A L V Y AN G S L C G G T L I N Q E W V L T A R H C D R G N M R I Y L G M H N L KV L N K D A L R R F P K E K Y F C L N T R N D T I W D K D I M L I R L NR P V R N S A H I A P L S L P S N P P S V G S V C R I M G W G T I T S PN A T L P D V P H C A N I N I L D Y A V C Q A A Y K G L A A T T L C A GI L E G G K D T C K G D S G G P L I C N G Q F Q G I L S V G G N P C A QP R K P G I Y T K V F D Y T D W I Q S I I S G N T D A T C P P

In accordance with porcine codon-usage bias (which is applicable tomost, if not all, mammalian cells), the following single stranded DNAwas synthesised <SEQ ID 17>: GTG ATC GGC GGC GAC GAG TGC AAC ATC AAC GAGCAC CGC TTC CTG GCC CTG GTG TAC GCC AAC GGC AGC CTG TGC GGC GGC ACC CTGATC AAC CAG GAG TGG GTG CTG ACC GCC CGC CAC TGC GAC CGC GGC AAC ATG CGCATC TAC CTG GGC ATG CAC AAC CTG AAG GTG CTG AAC AAG GAC GCC CTG CGC CGCTTC CCC AAG GAG AAG TAC TTC TGC CTG AAC ACC CGC AAC GAC ACC ATC TGG GACAAG GAC ATC ATG CTG ATC CGC CTG AAC CGC CCC GTG CGC AAC AGC GCC CAC ATCGCC CCC CTG AGC CTG CCC AGC AAC CCC CCC AGC GTG GGC AGC GTG TGC CGC ATCATG GGC TGG GGC ACC ATC ACC AGC CCC AAC GCC ACC CTG CCC GAC GTG CCC CACTGC GCC AAC ATC AAC ATC CTG GAC TAC GCC GTG TGC CAG GCC GCC TAC AAG GGCCTG GCC GCC ACC ACC CTG TGC GCC GGC ATC CTG GAG GGC GGC AAG GAC ACC TGCAAG GGC GAC AGC GGC GGC CCC CTG ATC TGC AAC GGC CAG TTC CAG GGC ATC CTGAGC GTG GGC GGC AAC CCC TGC GCC CAG CCC CGC AAG CCC GGC ATC TAC ACC AAGGTG TTC GAC TAC ACC GAC TGG ATC CAG AGC ATC ATC AGC GGC AAC ACC GAC GCCACC TGC CCC CCC

This single-stranded DNA was annealed to complementary oligonucleotidesto give a double-stranded molecule. Restriction sites are included ateither end of the double-stranded DNA, to which is ligated a CD4 anchorand a P-selectin signal sequence in a similar way to that describedabove. The resulting molecule was ligated, as before, into thepHβActpr-1gpt vector.

As an alternative DNA source, a snake cDNA library could be screened onthe basis of the known protein sequence.

14. Co-Expression of TFPI-CD4 and Hirudin-CD5 Causes Inhibition ofTF-Dependent and TF-Independent Clotting

Stable transfectants expressing both TFPI-CD4 and hirudin-CD4 weregenerated. As shown in FIG. 21A, the primary transfectants expressedvariable levels of hirudin and low levels of TFPI. Despite this modestexpression by the majority of transfectants, however, the procoagulantphenotype of these cells was significantly reduced compared to controls(FIG. 21B). The cell-surface presence of both anticoagulant molecules onIL-1α-activated IPEC markedly prolonged the time to clot plasma toapproximately 300 seconds, which is approaching the time taken forrecalcified human plasma to clot spontaneously. Blocking studies withanti-hirudin and anti-TFPI antibodies confirmed that the alteredphenotype of these double transfectants was due to specific inhibitionof coagulation by the expressed hirudin and TFPI.

It will be understood that the invention is described above by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

References (The Contents of which are Incorporated Herein)

-   Bach F H, Winkler H, Ferran C, Hancock W W, Robson S C. Delayed    xenograft rejection. Immunology Today 1996; 17(8); 379-384.-   Bajaj S P, Sabharwal A K, Gorka J, Birktoft J J. Antibody-probed    conformation transitions in the protease domain of human factor IX    upon calcium binding and zymogen activation: putative high-affinity    Ca²⁺-binding site in the protease domain. PNAS USA 1992; 89:    152-156.-   Bradley A, Liu P. Target practice in transgenics. Nature Genet.    1996; 14: 121-123.-   Clarke A R. The adenovirus and the egg: a new approach to    transgenesis. Nature Biotech 1996; 14: 942.-   Dang Q D, Guinto E R, Di Cera E. Rational engineering of activity    and specificity in a serine protease. Nature Biotech. 1997; 15:    146-149.-   Diamond L E, McCurry K R, Oldham E R, Tone M, Waldmann H, Platt J L,    Logan J S. Human CD59 expressed in transgenic mouse hearts inhibits    the activation of complement. Transplant Immunol. 1995; 3: 305-312.-   Disdier M, Morrissey J H, Fugate R D, Bainton D F, McEver R P.    Cytoplasmic domain of P-selectin (CD62) contains the signal for    sorting into the regulated secretory pathway. Molec. Biol. Cell.    1992; 3: 309-321.-   Dodt J, Kohler S, Baici A. Interaction of site specific hirudin    variants with alpha-thrombin. FEBS Lett. 1988; 229: 87-90-   Green S A, Setiadi H, McEver R P, Kelly R B. The cytoplasmic domain    of P-selectin contains a sorting determinant that mediates rpaid    degradation in lysosomes. J. Cell Biol. 1994; 124: 435-448.-   Gunning P, Leavitt J, Muscat G, Ng S Y, Kedes L. A human beta-actin    expression vector system directs high-level accumulation of    antisense transcripts. PNAS USA.1987; 84: 4831-5-   Hamamoto T, Yamamoto M, Nordfang O, Petersen J G L, Foster D C,    Kisiel W. Inhibitory properties of full-length and truncated    recombinant TFPI variants expressed in Saccharomyces cereviaiae. J.    Biol. Chem 1993; 268: 13344-13351.-   Heckl-Östreicher B, Binder R, Kirschfink M. Functional activity of    the membrane-associated complement inhibitor CD59 in a pig-to-human    in vtiro model for hyperacture xenograft rejection. Clin. Exp.    Immunol 1995; 102:589-595.-   Holmes N, Ennis P, Wan A M, Denney D W, Parham P. Multiple genetic    mechanisms have contributed to the generation of the HLA-A2/A28    family of class I MHC molecules. J Immunol. 1987; 139: 93641-   Johnston G I, Cook R G, McEver R P. Cloning of GMP-140, a granule    membrane protein of platelets and endothelium: sequence similarity    to proteins involved in cell adhesion and inflammation. Cell. 1989;    56:1033-44-   Kiely J-M, Cybulsky M I, Luscinskas F W, Gimborne M A.    Immunoselective targeting of an anti-thrombin agent to the surface    of cytokine-activated vascular endothelial cells. Arterioscier.    Thromb. Vasc. Biol. 1995; 15: 1211-1218.-   Kisiel A, Kondo S, Smith K J, McMullen B A, Smith L F.    Characterization of a protein C activator from Agkistrodon    contortrix contortrix venom. J. Biol. Chem. 1987;262:12607-13.-   Knapp A, Degenhardt T, Dodt J. Hirudisins. J. Biol. Chem. 1992; 34:    24230-24234.-   Langford G A, Cozzi E, Yannoutsos N, Lancaster R, Elsome K, Chen P,    White D G J. Production of pigs transgenic for human regulators of    complement activation using YAC technology. Transplant Proc. 1996;    28: 862-863.-   Lechler R I, Bal V, Rothbard J B, Germain R N, Sekaly R, Long E O,    Lamb J. Structural and functional studies of HLA-DR restricted    antigen recognition by human helper T lymphocyte clones by using    transfected murine cell lines. J. Immunol. 1988, 141: 3003-3009.-   McCurry K R, Diamond L E, Kooyman D L, Byrne G W, Martin M J, Logan    J S, Platt J L. Human complement regulatory proteins expressed in    transgenic swine protect swine xenografts from humoral injury.    Transplant Proc. 1996; 28: 758.-   McMullen B A, Fujikawa K, Kisiel W. Primary structure of a protein C    activator from Agkistrodon contortrix contortrix venom. Biochemistry    1989; 28: 674-679.-   Maddon P J, Littman D R, Godfrey M, Maddon D E, Chess L, Axel R. The    isolation and nucleotide sequence of a cDNA encoding the T cell    surface protein T4: a new member of the immunoglobulin gene family.    Cell. 1985; 42: 93-104-   Mao S S, Huang J, Welebob C, Neeper M P, Garsky V M, Shafer J A.    Identification and characterization of variants of tick    anticoagulant peptide with increased inhibitory potency toward human    factor Xa. Biochemistry 1995; 34: 5098-5103.-   Merkenschlager M, Altmann D M, Ikeda H. T cell alloresponses against    HLA-DQ and -DR products involve multiple epitopes on the CD4    molecule. Distinct mechanisms contribute to the inhibition of HLA    class II-dependent and -independent T cell responses by antibodies    to CD4. J. Immunol. 1990;145: 3181-7.-   O'Brien D P, Kemball-Cook G, Hutchinson A M, Martin D M, Johnson D    J, Byfield P G. Takamiya O, Tuddenham E G, McVey J H. Surface    plasmon resonance studies of the interaction between factor VII and    tissue factor. Biochemistry 1994; 33:14162-9.-   Reinherz E L, Kung P C, Goldstein G, Schlossman S F. Separation of    functional subsets of human T cells by a monoclonal antibody. PNAS    USA.1979; 76: 40614065-   Schlaeppi J M. Preparation of monoclonal antibodies to the    thrombin/hirudin complex. Thromb Res. 1991; 62: 459-470-   Skem T, Bischoff R, Jallat S, Dott K, Ali-Hadji D, Clesse D. Kieny M    P, Courtney M. Sulphation of hirudin in BHK cells. FEBS. 1990; 1:    36-38.-   Squinto S P. Xenogeneic organ transplantation. Curr. Opin. Biotech.    1996; 7: 641-645.-   Wagner D D. The Weibel-Palade body: the storage granule for von    Willebrand factor and P-selectin. Thrombosis & Haemostasis. 1993;    70: 105-110.-   Wheeler M B. Development and validation of swine embryonic stem    cells: a review. Reprod Fertil. Dec. 1994; 6:563-568.-   White D, Cozzi E, Langford G, Oglesby T, Wang M, Wright L,    Wallwork J. The control of hyperacute rejection by genetic    engineering of the donor species. Eye 1995; 9: 185-189.-   Wun T C, Kretzmer K K, Girard T J, Miletich J P, Broze G J. Cloning    and characterization of a cDNA coding for the lipoprotein-associated    coagulation inhibitor shows that it consists of three tandem    Kunitz-type inhibitory domains. J. Biol. Chem. 1988; 263: 6001-4-   Yannoutsos N, Langford G A, Cozzi E, Lancaster R, Elsome K, Chen P,    White D J G. Production of pigs transgenic for human regulators of    complement activation. Transplant Proc. 1995; 27: 324-325.

1. A protein comprising a region with anticoagulant activity and aregion which can anchor said protein to a cell membrane.
 2. A proteinaccording to claim 1 further comprising a targeting sequence whichprevents the protein from being constitutively expressed at the cellsurface.
 3. A protein according to claim 1 or claim 2, wherein theanticoagulant region comprises the sequence of a hirudin, a tissuefactor pathway inhibitor, a tick anticoagulant peptide, or a protein Cactivator.
 4. A protein according to claim 1 or claim 2, wherein theanchor region is a sequence capable of attaching the protein to a lipidbilayer.
 5. A protein according to claim 4, wherein the anchor regioncomprises the transmembrane sequence from a membrane protein.
 6. Aprotein according to claim 2, wherein said targeting sequence is asequence which can target a nascent polypeptide to a secretory granule.7. A protein according to claim 6 wherein said secretory granule doesnot fuse with the cell's plasma membrane until the cell is suitablystimulated.
 8. A protein according to claim 7, wherein said secretorygranule is a Weibel-Palade body.
 9. A protein according to claim 6wherein said targeting sequence is the cytoplasmic domain of P-selectin.10. A protein according to any one of the previous claims wherein saidanchor sequence is that of P-selectin.
 11. A polynucleotide encoding aprotein according to any one of the previous claims.
 12. A vectorcomprising a polynucleotide according to claim
 11. 13. A delivery systemcomprising a protein according to any one of claims 1 to 10, apolynucleotide according to claim 11, or a vector according to claim 12.14. A method of transfecting a cell with a vector according to claim 12.15. A cell transfected according to claim
 14. 16. Biological tissuecomprising a cell according to claim
 15. 17. An animal comprisingbiological tissue according to claim 16 and/or cell according to claim15.
 18. An animal according to claim 17, wherein said animal is atransgenic pig or sheep.
 19. A method of rendering a tissue or organsuitable for transplantation, comprising expressing a protein accordingto any one of claims 1 to 10 on the surface of endothelial cells in saidtissue or organ.
 20. A method of transplantation comprisingtransplanting biological tissue according to claim 16 from a donoranimal into a recipient animal.